Fructose absorption inhibitor

ABSTRACT

A fructose absorption inhibitor according to the present invention comprises a hydrolyzable tannin as an active ingredient. The hydrolyzable tannin preferably has a form composed of a gallic acid derivative and/or an ellagic acid derivative bound to a hydroxy group in glucose via an ester bond, and includes ellagitannin, gallotannin and so on.

TECHNICAL FIELD

The present invention relates to a fructose absorption inhibitor whichinhibits the process of absorbing fructose contained in foods and drinksinto the body through the small intestine to prevent and improvelifestyle-related diseases such as obesity, fatty liver, diabetes andthe like caused by fructose intake.

BACKGROUND ART

Obesity is a state in which fat accumulates excessively in the body. Oneof the causes of fat accumulation in the body is excess intake ofsaccharides (carbohydrates). In general, when saccharides contained infoods and drinks are taken into the body, saccharides are digested bydigestive enzymes, mainly converted into monosaccharides and absorbedinto the body through the intestinal tracts.

One kind of monosaccharides, glucose, is digested and absorbed, and thenmetabolized by group of enzymes called the glycolytic pathway. Since themetabolism is regulated at the step with phosphofructokinase among theseenzymes, the fat synthetic pathway is not immediately activated even ifa large amount of glucose is taken.

On the other hand, another kind of the monosaccharides, fructose, ismetabolized through a pathway bypassing the step withphosphofructokinase unlike glucose. Therefore, if fructose is taken in alarge amount, fructose rapidly proceeds to the fat synthetic pathway inthe liver and generated fat accumulates in adipose tissue. Accordingly,the usual intake does not cause problems on health and safety, butexcess intake of fructose increases the risk of causing pathologicalconditions such as obesity.

In addition, fructose has strong sweetness among monosaccharides and hassuch a property that its sweetness is enhanced as the temperaturedecreases. Therefore, fructose is widely used as a sweetener forprocessed foods such as chilled sweets and soft drinks containinghigh-fructose corn syrup (HFCS) and the consumption has been rapidlyincreasing in recent years.

It is well known that the excessive intake of such soft drinkscontaining high-fructose corn syrup has become a social problem in eachcountry. Also in Japan, mainly among young males, cases that are called“PET bottle syndrome” or “soft drink ketosis” have been increasing,wherein daily intake of 1 liter or more of soft drink causes ketosis orketoacidosis. Further, diabetes patients have increased concentration offructose in the blood or increased excretion of fructose in the urine.Especially, it has been reported that the high postprandialconcentration of fructose in the blood correlates with diabeticretinopathy (Non-patent Document 1), which reports excess intake offructose can be a cause of diabetic complications.

Under such social background, WHO (World Health Organization) recommendsthat the intake of saccharides added as sweeteners should not exceed 10%of the total energy intake (Non-patent Document 2). For example, in theUnited States, ADA (American Diabetes Association) warns against use offructose as an alternative to sucrose in a guideline for medical carefor diabetes. Also in Japan, Ministry of Health, Labour and Welfareraises an alarm over excess intake of fructose in “The Dietary ReferenceIntakes for Japanese”, 2010 edition, developed by Ministry of Health,Labour and Welfare.

Excess intake of fructose can be a factor causing various diseases otherthan those mentioned above. That includes enhancement of oxidativestress in vivo (Non-patent Document 2), glycation of protein (Non-patentDocuments 3 and 4), calcium deposits in the kidney (Non-patent Document5), hyperuricemia (Non-patent Document 6), initiation of insulinresistance (Non-patent Document 7), initiation of cardiovascular kidneydiseases (Non-patent Document 8), non-alcoholic fatty liver disease(NAFLD) (Non-patent Document 9) and the like.

Glycation of protein means generation of AGE (Advanced GlycationEnd-product) through reactions between saccharides and proteins in vivo.In the process of the reactions, cellular aging and denaturation ofprotein occur. Generated AGEs also react with surrounding proteins andthe like and promote denaturation of body tissues and the like. SinceAGE is involved in aging of capillary blood vessels and the like, AGE isregarded as one of the causes of cataract, decrease in kidney functionand the like. In particular, AGE is considered to be strongly related tothe onset and aggravation of complications caused by diabetesprogression. Fructose has a strong reducing character. Therefore, therehave been many reports that fructose has much greater glycation powerthan glucose. Methylglyoxal, a by-product in the process of fructosemetabolism, is also problematic as a precursor of AGE.

Incidentally, one kind of the saccharides, sugar (sucrose), isdecomposed into the glucose and fructose by amylolytic digestiveenzymes. When a large amount of sugar is taken, rapid absorption ofglucose leads to surge in the blood glucose level along with immediatesecretion of insulin. Insulin helps to promote the pathway to convertfructose and glucose into lipid, glycogen synthesis and glucose intakeby adipocytes. Therefore, medical importance is also placed on sugar asa substance causing obesity and the like.

However, sugar is the best as a sweetener considering taste, so a largeamount of sugar is consumed for juice, confectionary, cooking and thelike.

Accordingly, in cases where a large amount of sugar is taken, if theabsorption of glucose and fructose generated by digestion of sugar intothe body can be inhibited, the effect of reducing the total calorieintake can be expected. However, glucose is the most importantmonosaccharide for mammals biochemically and is the major energy sourcefor various tissues. In particular, the brain usually uses glucose as asole energy source. Therefore, it is problematic to strongly inhibitabsorption of glucose in view of safety.

On the other hand, as mentioned above, few roles are confirmed aboutfructose except for a role as a calorie source. Then, less importance isplaced on fructose than glucose nutritionally. Therefore, it can beconsidered that as a preventive measure against obesity and the like incases where saccharides are taken excessively, the best way is tospecifically inhibit the process of absorbing fructose through theintestinal tracts into the body.

As conventional knowledge, concerning substances which specificallyinhibit absorption of fructose into the body, an extract of eucalyptusleaves (Patent Document 1), several kinds of natural extracts (PatentDocument 2), and glyco-1,3-oxazolidin-2-ones and analogs thereof thathave been synthesized as analogs of fructose or sorbitol (Non-PatentDocument 10) have been reported.

However, these natural extracts have a weak inhibitory activity and needto be used in a large amount. In addition, there are problems that it isdifficult to maintain the quality constant since natural extracts are amixture and that natural extracts are hard to be added to foods, drinks,animal feed or the like due to their distinctive tastes. Syntheticsubstances cannot be used for foods and drinks, and safety needs to bestrictly verified if they are used for drugs. So it is very difficult toput them to practical use.

PRIOR ART DOCUMENTS Patent Documents

-   Patent Document 1: Japanese Unexamined Patent Application    Publication 2003-160504-   Patent Document 2: Japanese Unexamined Patent Application    Publication 2009-184992

Non-Patent Documents

-   Non-Patent Document 1: T. Kawasaki et al., Metabolism, 53, 583-588    (2004)-   Non-Patent Document 2: A. Cavarape et al., J. Endocrinol. Invest.,    24, 838-845 (2001)-   Non-Patent Document 3: Takahiro Kawasaki et al., Diabetes, 48,    419-421 (2005)-   Non-Patent Document 4: C. G. Schalkwijk et al., Diabetes Metab. Res.    Rev., 20, 369-382 (2004)-   Non-Patent Document 5: Takatoshi Esashi, SHOKUHIN EISEIGAKU ZASSHI,    35, 409-412 (1994)-   Non-Patent Document 6: X. Gao et al., Hypertension, 50, 306-312    (2007)-   Non-Patent Document 7: S. S. Elliott et al., Am. J. Clin. Nutr., 76,    911-922 (2002)-   Non-Patent Document 8: R. J. Johnson et al., Am. J. Clin. Nutr., 86,    899-906 (2007)-   Non-Patent Document 9: T. Kawasaki et al., J. Nutr., 139, 2067-2071    (2009)-   Non-Patent Document 10: J. Girniene et al., Carbohyd. Res., 338    (2003)

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

Accordingly, it is desirable to provide a fructose absorption inhibitorwhich has rich history of use in foods with high safety and exerts asufficient effect with a small dosage for adding it to various foods,drinks and animal feed and for using it as a drug. Further, stable andinexpensive availability in a large amount of such a fructose absorptioninhibitor is industrially very useful.

An object of the present invention is to provide a fructose absorptioninhibitor which is derived from natural products, able to specificallyinhibit absorption of fructose into the body, safe, applicable tovarious foods, drugs and animal feed, and easy to take.

Means for Solving the Problems

The present inventors intensively studied to solve the problems. As aresult, they found that a hydrolyzable tannin inhibits fructoseabsorption through the intestinal tracts very strongly, leading to thepresent invention. The hydrolyzable tannin has an ester bond composed ofa hydroxyl group in a monosaccharide such as glucose and an ellagic acidderivative and/or a gallic acid derivative.

That is, a fructose absorption inhibitor of the present inventionincludes the following configurations.

(1) A fructose absorption inhibitor containing a hydrolyzable tannin asan active component.

(2) The fructose absorption inhibitor as set forth in (1), wherein thehydrolyzable tannin is a compound having an ester bond composed of ahydroxy group in glucose and a gallic acid derivative and/or an ellagicacid derivative.

(3) The fructose absorption inhibitor as set forth in (1) or (2),wherein the hydrolyzable tannin is ellagitannin having an ester bondcomposed of hydroxy groups at the 2- and 3-positions or the 4- and6-positions or at the both positions in glucose and at least onehexahydroxydiphenoyl group or valoneoyl group.

(4) The fructose absorption inhibitor as set forth in (1) or (2),wherein the hydrolyzable tannin is gallotannin having an ester bondcomposed of at least three hydroxy groups in glucose and gallic acids.

Effect of the Invention

The fructose absorption inhibitor of the present invention has a highfructose absorption inhibitory effect in the intestinal tracts.Therefore, the fructose absorption inhibitor is effective to prevent,improve and treat obesity and various diseases caused by excess intakeof fructose. Moreover, the hydrolyzable tannin used in the presentinvention is often contained in natural products used for foods and thelike, which also ensures high safety. Further, the hydrolyzable tanninsused in the present invention are easy to be taken or administered, andthe effect can be expected with a small amount of usage as well.Therefore, the fructose absorption inhibitor of the present inventioncan be contained in various foods, drugs, animal feed and the like. Thenvarious foods, drugs, animal feed and the like suitable for intake oradministration can be produced.

MODE FOR CARRYING OUT THE INVENTION

The fructose absorption inhibitor of the present invention contains ahydrolyzable tannin as an active component. The fructose absorptioninhibitor may also be contained in foods, drinks, animal feed, quasidrugs or drugs.

A hydrolyzable tannin is a kind of polyphenols and has an ester bondcomposed of a polyol such as glucose and a gallic acid derivative and/oran ellagic acid derivative. In the present invention, the hydrolyzabletannin preferably has an ester bond composed of glucose and a gallicacid derivative and/or an ellagic acid derivative and the glucose may bering-opened.

The gallic acid derivatives include, as represented by the followinggeneral formula (1), compounds having the gallic acid structure as abasic skeleton with a hydroxy group in a gallic acid substituted with analkyl group, an acyl group or the like.

[wherein R¹ represents a hydroxy group or an alkoxy group and R², R³ andR⁴, which are the same or different, each represent a hydrogen atom, analkyl group or an acyl group.]

The gallic acid derivative may be an oligomer such as a dimer or atrimer in which several structures represented by the general formula(1) are bound to each other via ether bond or ester bond; compounds inwhich a structure represented by the general formula (1) is bound to oneor more ellagic acid derivatives via an ether bond or ester bond; andthe like.

Examples of the ellagic acid derivatives include compounds having theellagic acid structure as a basic skeleton with its hydroxy groupsubstituted with an alkyl group, an acyl group or the like. For example,they are represented by the following general formula (2). In addition,a compound having a hexahydroxydiphenoyl (hereinafter referred to as“HHDP” for short) group derived from an oxide of an ellagic acid and acompound having a valoneoyl group in which a gallic acid is added to anHHDP group are also included in ellagic acid derivatives.

[wherein R¹, R², R³ and R⁴, which are the same or different, eachrepresent a hydrogen atom, an alkyl group or an acyl group.]

The ellagic acid derivative may be compounds in which a structurerepresented by the general formula (2) is bound to one or more gallicacid derivatives represented by the general formula (1) via an etherbond or ester bond; an oligomer such as a dimer or a trimer in whichseveral structures represented by the general formula (2) are bound toeach other via ether bond or ester bond; and the like.

As the hydrolyzable tannin, both of ellagitannin and gallotannin may bepreferably used.

Ellagitannin refers to compound groups having an ester bond composed ofa hydroxy group in a polyhydric alcohol such as glucose and an HHDPgroup derived from an oxide of an ellagic acid, and an ellagic acid anda polyhydric alcohol are generated by hydrolysis.

Gallotannin refers to compound groups having an ester bond composed of ahydroxy group in a polyhydric alcohol and a gallic acid, and a gallicacid and a polyhydric alcohol are generated by hydrolysis.

More preferably, the hydrolyzable tannins include, for example,ellagitannin having an ester bond composed of hydroxy groups at the 2-and 3-positions or the 4- and 6-positions or at the both positions inglucose and at least one HHDP group or valoneoyl group; gallotanninhaving an ester bond composed of at least three hydroxy groups inglucose and gallic acids; and the like.

As used herein, a valoneoyl group is a group having an ether bondcomposed of an HHDP group and a galloyl group illustrated in thefollowing structure (3). Examples of the ellagitannin also include manycompounds having an ester bond composed of other hydroxy group in apolyhydric alcohol and a gallic acid.

Specific examples of the hydrolyzable tannin include, as illustrated inthe following structures (4) to (8), Tellimagrandin I and II,Strictinin, Casuarictin, 1,3-di-O-galloyl-4,6-HHDP-β-D-glucose,Oenothein B, Eugeniflorin D₂, 1,2,3-trigalloyl glucose,1,2,3,6-tetragalloyl glucose, 1,2,3,4,6-pentagalloyl glucose and thelike. Among these, the hydrolyzable tannin is desirably at least oneselected from the group consisting of Tellimagrandin I and II,1,3-di-O-galloyl-4,6-HHDP-β-D-glucose, Oenothein B and Eugeniflorin D₂,or at least one selected from the group consisting of 1,2,3-trigalloylglucose, 1,2,3,6-tetragalloyl glucose and 1,2,3,4,6-pentagalloylglucose.

These are compounds illustrated in the following structural formulae (4)to (8). Note that G in the following structural formulae (4) to (8)represents the following structure (3).

Examples of the raw materials for the hydrolyzable tannin include, butare not particularly limited to, Archichlamydeae belonging toAngiospermae Dicotyledoneae (Engler system) containing a hydrolyzabletannin, a tannic acid and the like.

A method for producing the hydrolyzable tannin having fructoseabsorption inhibitory activity from the plants is not particularlylimited and the hydrolyzable tannin may be produced by commonly usedmethods. In addition, when the hydrolyzable tannin is obtained byextraction, there are no specific limitations to the extractionconditions. For example, various parts of the plants (whole plant,flower, calyx, seed, fruit, leaf, branch, bark, root bark, rhizome, rootand the like) may be squeezed or extracted by a solvent as they are orafter cut, ground or pulverized to obtain an extract of the hydrolyzabletannin.

Examples of the plants include Myrtaceae, Rosaceae, Casuarinaceae,Fagaceae, Theaceae, Onagraceae, Lythraceae, Trapaceae, Punicaceae,Melastomataceae, Combretaceae, Lecythidiaceae and the like. Since theseplants contain the hydrolyzable tannin in a large amount, thehydrolyzable tannin may be efficiently obtained by using these plants asa raw material. Among these, Myrtaceae plants are preferable. Further,plants belonging to Eucalyptus, Syzygium, Pimenta and Melaleuca areoften used for foods, spices, flavors and the like. Therefore, it ispreferable to use as a raw material eucalyptus, clove, allspice or thelike, all of which belong to these genera, from a viewpoint of historyof use in foods and safety. Especially, eucalyptus is preferable becauseeucalyptus has a very strong fructose absorption inhibitory activity,and an extract of eucalyptus leaves contains an affluent amount of thehydrolyzable tannin and contains Tellimagrandin I and II, Oenothein B,galloyl glucoses and the like in a large amount.

Among these, extraction using a solvent is performed under suchconditions that the hydrolyzable tannin is eluted. For example,depending on the solvent used, extraction may be performed under theconditions of normal to increased pressure and a temperature from roomtemperature to the boiling point of the solvent for around 10 minutes to1 week.

As the solvent used for extraction, solvents commonly used may beselected to use as appropriate according to the kind of plants andtreatment processes. Examples of the solvents include water; organicsolvents such as alcohols (for example, lower alcohols such as methanoland ethanol, or polyhydric alcohols such as ethylene glycol, propyleneglycol, 1,3-butylene glycol and glycerin); ketones having relativelyhigh polarity such as acetone; esters such as ethyl acetate, and thelike. Among these, solvents combining methanol, ethanol or acetone withwater are preferable. When the residue of an organic solvent is notpreferable as in cases of being used as foods, it is especiallypreferable to use water, ethanol and aqueous ethanol. These solvents maybe used solely or any two or more kinds thereof may be used incombination.

There are no specific limitations to the method for extracting thehydrolyzable tannin, and homogenizing extraction at room temperature,reflux extraction, supercritical fluid extraction and the like may beused.

For example, the following method may be used. An intact plant materialor a dried plant material containing a large amount of the hydrolyzabletannin is pulverized. Then, 5 to 20-fold amount of an extraction solventbased on the total amount of the intact plant material or dried plantmaterial is added to the pulverized plant. The mixture is allowed tostand under normal pressure at room temperature for around 1 week, or isextracted at around the boiling point of the extraction solvent foraround 10 to 30 minutes. After that, the filtrate obtained by filtrationis dried under reduced pressure or freeze-dried to obtain a plantextract.

The plant extract obtained as mentioned above may be used as it is,since the extract contains a large amount of the hydrolyzable tannin.Further, if needed, a purification treatment such as deodorization ordecolorization may be additionally performed as long as the treatmentdoes not affect the hydrolyzable tannin.

Any usual method may be selected to carry out the method for suchpurification treatments. For example, filtration, liquid-liquidextraction, ion-exchange resin, activated carbon column or the like maybe used for adsorption, decolorization, purification and the like.Further, a purified product in a form of solution, paste, gel or powdermay be obtained by freeze drying, a concentration treatment or the like.

There are no specific limitations to the form of the fructose absorptioninhibitor of the present invention. For example, the fructose absorptioninhibitor may be used in a unit dosage form which contains thepredetermined amount of the hydrolyzable tannin in a desiredformulation, or the extract or purified product obtained from plantsusing the above-described method may also be used as it is. When used ina unit dosage form, for example, it may be used as a compositioncontaining the fructose absorption inhibitor and other components addedas necessary.

Examples of such compositions include a composition containing thefructose absorption inhibitor and a suitable carrier (such as a carrierwhich is used for foods or drugs), and a composition containing thefructose absorption inhibitor and fructose.

There are no specific limitations to the formulation of the fructoseabsorption inhibitor. For example, any forms suitable for use as foods(food and drink), drugs, animal feed, additives for animal feed and thelike may be used.

The content of the hydrolyzable tannin in the fructose absorptioninhibitor of the present invention may be preferably 1 to 5000 mg, morepreferably 10 to 3000 mg and especially preferably 50 to 1000 mg perunit dose of the fructose absorption inhibitor. When the content of thehydrolyzable tannin per unit dose of the fructose absorption inhibitoris less than 1 mg, there is a worry that the high fructose absorptioninhibitory effect in the intestinal tracts is insufficient. On the otherhand, when the content exceeds 5000 mg, there is a worry that an effectwhich meets the content of the hydrolyzable tannin cannot be obtained.In addition, intake of more hydrolyzable tannin than necessary can causediarrhea depending on a person's constitution.

The unit dose refers to the predetermined amount calculated such thatthe fructose absorption inhibitory effect is exerted when the fructoseabsorption inhibitor of the present invention is taken in a form oftablets or other forms, in which form the amount is contained.

For providing a form of food, the hydrolyzable tannin is mixed with foodmaterials to prepare a form of, for example, solid food, creamy orjam-like semi-liquid food, gel-like food, drink or the like. When usedin such a form of food, especially use of fructose and/or thepolysaccharides containing fructose in combination makes it possible toproduce foods which are excellent in palatability and also can inhibitabsorption of fructose.

When using the fructose absorption inhibitor in a form of food, variouscomponents commonly used for foods may be contained. Examples of suchcomponents include, glucose, maltose, sorbitol, stevioside, corn syrup,lactose, citric acid, tartaric acid, malic acid, succinic acid, lacticacid, L-ascorbic acid, dl-α-tocopherol, glycerin, propylene glycol,glycerin fatty acid ester, polyglycerin fatty acid ester, sucrose fattyacid ester, sorbitan fatty acid ester, propylene glycol fatty acidester, gum arabic, carrageenan, casein, gelatin, pectin, agar, vitamin Bcomplex, nicotinic acid amide, calcium pantothenate, amino acids,calcium salts, food colorings, flavors, preservatives and the like.These may be blended depending on the kind of foods as appropriate.

Specific examples of the foods include soft drink, juice, coffee, tea,liqueur, milk, whey beverage, lactic fermenting beverage, candy, chewinggum, chocolate, gummi, yoghurt, ice cream, pudding and the like.Addition of the extract or the fructose absorption inhibitor to foods issuitably carried out by adding the fructose absorption inhibitor so thatthe content of the hydrolyzable tannin is from 0.5 to 100 mg/g. In casesof supplements, there are no problems in safety and effect even if it iscontained 90% by weight.

For use in a form of the pharmaceutical preparation, the hydrolyzabletannin is mixed with a pharmaceutically-acceptable common carrier toprepare a form of solid, semi-solid or liquid. Specific forms include,for example, oral administration agents such as tablets, capsules,pills, granules, powders, emulsions, suspensions, syrups and pellets,parenteral administration agents such as suppositories, and the like.

In formulating, conventionally used carriers such as surfactants,excipients, binders, disintegrant, lubricants, preservatives,stabilizers, buffers, suspensions may be used depending on theformulation. Preferably, examples of the carriers include, solidcarriers such as starch, lactose, mannitol, carboxymethylcellulose, cornstarch and inorganic salts; liquid carriers such as distilled water,physiological saline, an aqueous solution of glucose, alcohols (such asethanol), propylene glycol and polyethylene glycol; oily carriers suchas various animal and vegetable oils, white petrolatum, paraffin andwax; and the like.

Since the pharmaceutical preparation contains the hydrolyzable tannineffective for inhibiting fructose absorption as an active component, ithas a fructose absorption inhibitory effect. Therefore, thepharmaceutical preparation is effective for prevention, improvement andtreatment of various disorders and diseases caused by excess intake offructose.

For example, the pharmaceutical preparation may be applied to use aspreventive or therapeutic agents against diseases such as enhancement ofoxidative stress in vivo, glycation of protein, calcium deposits in thekidney, hyperuricemia, ketosis, initiation of insulin resistance,initiation of cardiovascular kidney diseases and diabetic complication(such as diabetic kidney dysfunction and cataract, and such as necrosisof lower extremities) as well as hyperlipidemia, simple fatty liver andnonalcoholic fatty liver disease. In addition to that, thepharmaceutical preparation may also be applied to use as anti-obesityagents, fat accumulation inhibitors for visceral fat, subcutaneous fatand the like, anti-arteriosclerosis agents, thrombus prevention agents,hypotriglyceridemic agents, agents for lowering blood cholesterol levelsand the like.

When producing animal feed which inhibits fructose absorption by usingthe hydrolyzable tannin in the present invention, one or two or morekinds of the extracts are mixed with various components used for animalfeed for preparation.

In addition, the hydrolyzable tannin in the present invention may beused in a form of an additive for animal feed. In this case, the extractmay be added to animal feed as it is. Alternatively, the additive foranimal feed may be prepared to have a form of powder, granules,capsules, syrup, gel, liquid, solid or the like. Examples of the animalfeed to which the additive for animal feed is added include the kinds ofanimal feed as mentioned above. The added amount may be the same levelas the blended amount of the animal feed as mentioned above.

The animal feed may be added at any steps during production or afterproduction.

EXAMPLES

Hereinafter, the present invention will be described specifically withreference to examples, but the present invention is not limited to thefollowing examples. Preparation of the extract and hydrolyzable tanninused in examples, evaluation of fructose absorption inhibitory activity,and handling of measured data were carried out by the following methods.

<Method for Preparing Extract> (Crude Extract of Eucalyptus Leaves)

First of all, 5 kg of eucalyptus leaves was refluxed for 2 hours in 45kg of 30% ethanol. Then, after cooled to room temperature, the resultantmixture was filtrated. The obtained filtrate was subjected to vacuumconcentration and freeze drying to obtain a crude extract of eucalyptusleaves (yield: about 1 kg).

(20% Ethanol-Eluted Fraction)

To a resin (“DIAION (registered trademark) HP20” manufactured byMitsubishi Chemical Corporation), 100 g of the resultant crude extractof eucalyptus leaves was adsorbed. Elution was then carried outsequentially with 0 to 100% ethanol to obtain each eluate. And eachfractionated eluate was subjected to vacuum concentration and freezedrying. Among these, the 20% ethanol-eluted fraction was found to have astrong fructose absorption inhibitory activity.

(60% Methanol-Eluted Fraction)

Regarding the 20% ethanol-eluted fraction among the resultant eacheluate, 5 g out of 16 g of yield was adsorbed to a resin (“TOYOPEARL(registered trademark) HW40 (f grade)” manufactured by TOSOHCORPORATION), and elution was carried out sequentially using 40 to 100%methanol as an eluent. High performance liquid chromatography was usedfor component analysis of each eluate. That is, while the solventconcentration was changed in the gradient mode, 5% aceticacid-acetonitrile (100%→0%) was flowed to a PAQ column (manufactured byNACALAI TESQUE, INC.) at 40° C., and component distribution of eacheluted fraction was monitored with the measuring range of a photodiodearray detector set at 270 to 350 nm. About 20 fractions were obtained byfractionation. Among these, the 60% methanol-eluted fraction was foundto have a strong fructose absorption inhibitory activity.

(Tellimagrandin I)

Regarding the 60% methanol-eluted fraction among each eluted fractionobtained by fractionating the 20% ethanol-eluted fraction, 80 mg out of106 mg of yield was repeatedly subjected to HPLC fractionation by a PAQcolumn using the same eluent as mentioned above to obtain TellimagrandinI (32 mg). Identification of the substance was confirmed by comparingthe retention time of HPLC and various NMR data with those of thestandard substance.

As other hydrolyzable tannins, those isolated and identified fromvarious Myrtaceae plants such as eucalyptus leaves were used.

<Evaluation Method of Fructose Absorption Inhibitory Activity in theIntestinal Tracts>

Evaluation of fructose absorption inhibitory activity was carried outusing the human colon carcinoma cell line, Caco-2 (manufactured byDainippon Sumitomo Pharma Co., Ltd.) utilized for model experiments ofthe small intestinal mucosa. Medium was prepared for use by adding FCS(manufactured by BIOWEST) and NEAA (manufactured by SIGMA) to DMEMmedium (manufactured by SIGMA) such that the content of FCS is 10% andthat of NEAA is 1%.

Caco-2 cells were seeded in a 6-well Transwell Insert (internal area:4.2 cm²) and subcultured for around 3 weeks, while DMEM medium wasreplaced once every 3 to 4 days, until the Caco-2 cell density becamearound 2×10⁵ cells/insert. The TEER value was measured beforeexperiments and the state of Caco-2 cells was confirmed. Each sampleillustrated in Table 1 was dissolved in 10% dimethyl sulfoxide (DMSO).

Medium in the 6-well Transwell Insert was removed. After both of theinside and outside of the insert were washed with a phosphate buffersolution (pH 7.2) (PBS), D-PBS (manufactured by GIBCO) which did notcontain saccharides and serum was added to the insert. After 30 min ofincubation at 37° C. under 5% CO₂, the TEER value was measured. A samplesolution of each sample illustrated in Table 1 was added to the insertand each mixture was preincubated for 5 min. A fructose solution wasadded such that the final concentration of fructose is 50 mM and eachmixture was incubated at 37° C. under 5% CO₂ for 3 h. The TEER valueswere measured. Only for ones whose TEER values did not reduce after theculture, a permeated solution outside the insert was collected andstored at −80° C. The measurement was carried out three times for eachsample illustrated in Table 1.

The fructose concentrations of the permeated solutions were measured byan enzymatic method using D-fructose dehydrogenase (derived fromGluconobac sp.) (The concentration of each reagent represents the finalconcentration, respectively.). That is, to a mixed solution containing100 mM PBS (pH 6.0), 1% Triton X-100, 0.2 mM WST-1, 8 μM 1-methoxy PMSand 10 U fructose dehydrogenase (manufactured by TOYOBO CO., LTD.), thepermeated solution was added. The resulting mixture was reacted at 30°C. for 3 h, followed by measurement of the absorbance at 438 nm.

The fructose absorption inhibitory rate of each compound was determinedusing the following formula (1). Results are illustrated in Table 1. Ascontrol, measurement was also carried out for gallic acid, ellagic acid,quercetin, (+)-catechin, (−)-epicatechin and (−)-epigallocatechingallate.

(Permeated amount of fructose for blank (10% DMSO)−Permeated amount offructose for sample)/Permeated amount of fructose for blank×100  (I)

TABLE 1 Sample Dosage Inhibitory No. *1 Name of Samples (μg/ml) Rate (%)1 Tellimagrandin I 5 49 2 Tellimagrandin II 5 52 3 Strictinin 5 43 4Casuarictin 5 46 5 1,3-di-O-galloyl-4,6-HHDP-β-D-glucose 5 57 6Eugeniflorin D₂ 5 53 7 Oenothein B 5 63 8 1,2,3,4,6-pentagalloyl glucose5 53 9 1,2,3,6-tetragalloyl glucose 5 50 10 1,2,3-trigalloyl glucose 561 * 11 Gallic acid 50 19 * 12 Ellagic acid 50 22 * 13 Quercetin 50 23 *14 (+)-catechin 50 <10 * 15 (−)-epicatechin 50 <10 * 16(−)-epigallocatechin gallate 5 28 *1: Comparative Examples are indicatedwith “*”.

As illustrated in Table 1, the hydrolyzable tannins of samples of No. 1to 10 had 43% or more of the inhibitory rate, even though the dosage wasonly 5 μg/mL. That illustrates the hydrolyzable tannins of samples ofNo. 1 to 10 had a strong fructose absorption inhibitory activity. Fromthis result, it is expected that the hydrolyzable tannin inhibitsabsorption of fructose into the body in the small intestine andsuppresses obesity and the like caused by excess intake of fructose.Examples illustrate that the dosage of 5 μg/mL of the hydrolyzabletannin is effective in the absorption model experiments using theintestinal epithelial cell, Caco-2 (surface area: 4.2 cm²). However, inmammals including humans, intestinal epithelial cells to absorb fructoseexist in a far greater number than those in this absorption modelexperiments, and its surface area is estimated to reach 200 m² inhumans. Therefore, clinically, the effective content of the hydrolyzabletannin in the present invention is considered to be at least 1 mg,preferably 10 mg or more, and more preferably 50 mg or more.

On the other hand, in the case where the crude extract of eucalyptusleaves (corresponding to the extract in Patent Document 1) was used, thedosage of 1000 μg/mL led to the inhibitory rate of 65% and the dosage of100 μg/mL led to the inhibitory rate of less than 20%. Thus, in order toexert fructose absorption inhibitory activity by using the crude extractof eucalyptus leaves as it is, the dosage of 1000 μg/mL is needed.

The above-described Examples do not limit the present invention and areof course applicable within a scope without departing from the spiritsof the present invention. The above-described Examples explain only onecomponent obtained from eucalyptus leaves (Tellimagrandin I). However,not only eucalyptus but also other plants containing the hydrolyzabletannin may be used, and they may be partly purified for use as well.Further, a mixture of the hydrolyzable tannins may also be used.

1. A fructose absorption inhibitor comprising a hydrolyzable tannin as an active component.
 2. The fructose absorption inhibitor according to claim 1, wherein the hydrolyzable tannin is a compound having an ester bond composed of a hydroxyl group in glucose and one or more of a gallic acid derivative or an ellagic acid derivative.
 3. The fructose absorption inhibitor according to claim 2, wherein the gallic acid derivative is a compound represented by the general formula (1):

wherein R¹ represents a hydroxy group or an alkoxy group, and R², R³ and R⁴, which are the same or different, each represent a hydrogen atom, an alkyl group or an acyl group.
 4. The fructose absorption inhibitor according to claim 2, wherein the ellagic acid derivative is a compound represented by the general formula (2), a compound having a hexahydroxydiphenoyl group derived from an oxide of an ellagic acid, or a compound having a valoneoyl group in which a gallic acid is added to a hexahydroxydiphenoyl group:

wherein R¹, R², R³ and R⁴, which are the same or different, each represent a hydrogen atom, an alkyl group or an acyl group.
 5. The fructose absorption inhibitor according to claim 1, wherein the hydrolyzable tannin is ellagitannin having an ester bond composed of hydroxy groups at the 2- and 3-positions or the 4- and 6-positions or at the both positions in glucose and at least one hexahydroxydiphenoyl group or valoneoyl group.
 6. The fructose absorption inhibitor according to claim 1, wherein the hydrolyzable tannin is gallotannin having an ester bond composed of at least three hydroxy groups in glucose and gallic acids.
 7. The fructose absorption inhibitor according to claim 1, which has a form of food or drug.
 8. The fructose absorption inhibitor according to claim 1, which has a form of animal feed or an additive for animal feed.
 9. The fructose absorption inhibitor according to claim 1, wherein the hydrolyzable tannin is at least one selected from the group consisting of Strictinin, Casuarictin, Oenothein B, 1,2,3-trigalloyl glucose and Eugeniflorin D₂. 